The primary objective of this proposal is to measure the extent to which microregional heterogeneities in tumor vascular structure and oxygen delivery relate to differences in radiotherapeutic response. Comparing two tumor lines of differing radiobiological hypoxic fraction (HF), RIF-1 and KHT, our preliminary work has revealed substantial interline differences in: 1) vascular geometry, 2) oxygen availability, and 3) response to oxygen manipulation. Within a given tumor line, increases in tumor volume and HF were shown to correspond closely with decreases in overall HbO2 levels. Following improvements in sampling techniques, microvessel resolution, and spatial analysis, inter-tumor line correlations between HF and HbO2 levels also appeared promising. We propose to further refine our techniques by quantifying the significance and prevalence of such complicating factors as intermittent flow, necrotic distribution, and distribution of diffusion distances. Recently derived techniques, using cryospectrophotometry, dual fluorescent labeling, and immunofluorescence, will allow us to measure each of these parameters on consecutive tumor slices. This will provide a quantitative characterization of the physiological relationships between tumor oxygen supply, acute vs. chronic hypoxia, and development of necrosis. Once these basic relationships are established, we will focus on changes following single-dose and fractionated radiotherapy. Pronounced differences in reoxygenation have previously been observed in these lines, and our aim will be to define changes in microregional oxygen availability, vascular geometry, and flow distribution following treatment. Our next goal will be to evaluate tumor response to both hemodynamic (blood pressure/flow) and rheologic (blood viscosity) manipulation. Diverse responses between lines, such as observed with flunarizine, should not only provide detailed information regarding the efficacy of individual agents at the local level, but also important clues as to the underlying functional differences between lines. This will provide a level of understanding not attainable using previously available volume-averaged measurements such as HF and overall flow. Finally, an existing mathematical model will be utilized and refined on the basis of our increasing understanding of heterogeneous vascular geometry, oxygen distribution, and intermittent flow. This will allow us to gauge the relative effects of the different parameters and will also provide a concise theoretical representation of the physiologic processes relating to both reoxygenation and oxygen manipulation. Microregional changes measured using these methodologies will also be valuable in interpreting results obtained using alternative invasive and noninvasive techniques including laser Doppler flowmetry, oxygen electrodes, 31P-NMR spectroscopy, hypoxia markers, and Xenon clearance.